Project Summary/Abstract The three-dimensional (3D) organization of the genome affects many genomic functions. Multiple 3D genome architectures at different length scales, including chromatin loops, domains, compartments, and regions associated with nuclear lamina and nucleoli, have been discovered. Changes in these architectures have been associated with normal development, aging, and a wide range of diseases. However, how these structures are arranged in the same cell, how they vary in different cell types in mammalian tissue, and how they are correlated with gene expression and the epigenome in the tissue contexts are largely unknown. Current approaches are severely limited by a lack of capacity to simultaneously trace chromatin folding across multiple length scales, measure genomic organization in relation to other nuclear components, and profile gene expression and epigenome in the same single cells in mammalian tissue. To address this need, here we propose to develop a new methodology, termed Multiplexed Imaging of Nucleome Architectures (MINA), which will enable simultaneous measurements of multiscale chromatin folding, associations of genomic regions with nuclear lamina, nucleoli and other nuclear/epigenetic components, and copy numbers of numerous RNA species in the same, single cells in mammalian tissue. We expect this development to broadly impact many lines of research on 3D genomics by depicting cell-type-specific multiscale genomic architectures associated with gene expression and epigenome, in different types of tissue undergoing different biological processes. Furthermore, by allowing measurements within the same cell, it will in future be possible to directly test the causal relationship between genomic architecture and gene expression, thus opening up a completely new experimental paradigm to identify novel mechanisms that regulate gene expression across human development, health and disease.